The long-term goal of this project is to gain a better understanding of the molecular pathogenesis of urinary stone disease or urolithiasis. A centrally important function in maintaining urinary system homeostasis is to prevent supersaturated urine from forming crystals. Clearly a powerful set of defense mechanisms is required, but these mechanisms have been only partially characterized to date. During the last granting period, we have focused on in vivo roles of Tamm-Horsfall protein (THP, or uromodulin) in urinary tract defense. We found that inactivation of the THP gene predisposes mice to bladder colonization by transurethrally inoculated type 1-fimbriated E. coli. In addition, mice lacking THP develop spontaneous and chemically induced renal calcium crystals. The induction of renal crystals is accompanied by a marked increase in renal epithelial cells of osteopontin (OPN), a potent inhibitor of bone mineralization, vascular calcification and renal stone formation, raising the possibility that THP and OPN can act synergistically in inhibiting renal crystallization. In the next granting period, we will gain deeper insights into the role of THP as a critical innate defense factor in the urinary system by focusing on its role in preventing renal crystallization. First, we will examine whether THP knockout and THP mutation have different consequences on renal uric acid handling, by either inhibiting/inactivating uricase in THP knockout mice or by transgenically expressing human-relevant THP mutants in renal epithelial cells under the direction of the THP promoter. Second, we will examine whether the protective role of THP against renal crystallization is due directly to its modulation on renal calcium level or due indirectly to its protection against renal epithelial injury. Third, we will test the hypothesis that THP and OPN are co-inhibitors of renal calcification, by generating THP/OPN double knockouts and comparing the severity of renal calcification with that in the single knockouts. Finally, we will assess gene expression alterations in the renal epithelial cells in response to THP deficiency in order to better understand the roles of THP and other macromolecules in modulating renal crystallization and other renal functions. Results from these studies will shed new light on the molecular pathogenesis and intervention strategies for important urinary tract diseases such as urolithiasis, which afflicts millions of people annually in the United States alone.